Optimization of phenolics and flavonoids extraction conditions of Curcuma Zedoaria leaves using response surface methodology

This study focused on maximizing the extraction yield of total phenolics and flavonoids from Curcuma Zedoaria leaves as a function of time (80–120 min), temperature (60–80 °C) and ethanol concentration (70–90 v/v%).

RESEARCH ARTICLE

Optimization of phenolics

and flavonoids extraction conditions

of Curcuma Zedoaria leaves using response

surface methodology

Nur Fauwizah Azahar1,2, Siti Salwa Abd Gani1,2* and Nor Fadzillah Mohd Mokhtar2,3

Abstract

This study focused on maximizing the extraction yield of total phenolics and flavonoids from Curcuma Zedoaria leaves

as a function of time (80–120 min), temperature (60–80 °C) and ethanol concentration (70–90 v/v%) The data were subjected to response surface methodology (RSM) and the results showed that the polynomial equations for all mod-els were significant, did not show lack of fit, and presented adjusted determination coefficients (R2) above 99%, prov-ing their suitability for prediction purposes Usprov-ing desirability function, the optimum operatprov-ing conditions to attain a higher extraction of phenolics and flavonoids was found to be 75 °C, 92 min of extraction time and 90:10 of ethanol concentration ratios Under these optimal conditions, the experimental values for total phenolics and flavonoids of

Curcuma zedoaria leaves were 125.75 ± 0.17 mg of gallic acid equivalents and 6.12 ± 0.23 mg quercetin/g of extract, which closely agreed with the predicted values Besides, in this study, the leaves from Curcuma zedoaria could be

con-sidered to have the strong antioxidative ability and can be used in various cosmeceuticals or medicinal applications

Keywords: Curcuma zedoaria, Antioxidant activity, Response surface methodology, Phenolic, Flavonoids

© The Author(s) 2017 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/ publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.

Background

Plants are a substantial source of natural antioxidants

Active compounds present in natural antioxidants such

as phenolic, carotenoids, flavonoids, folic acid, benzoic

acid, and tocopherol are secondary metabolites of the

plants which can provide various potential treatment and

prevention of cancer, cardiovascular diseases,

neurode-generative diseases and etc [1 2]

Phenolics or polyphenols, including flavonoids, have

received greater attention because they are often

iden-tified as biological response modifiers and have

vari-ous functions such as metal chelators and free radical

terminators [3 4] The bioactive compounds present

in these compounds provide a variety of

physiologi-cal functions, for instance, antimicrobial, antiallergenic,

anti-inflammatory, and antimutagenic effects [5] More-over, it has been reported that the active compounds found in phenolic acids (caffeic, chlorogenic acid, ben-zoic acid) and flavonoids (catechin, quercetin, rutin) are potent antioxidants because they have all the right struc-tural features for free radical scavenging activity [6 7]

Curcuma zedoaria (Christm.) Roscoe from

Zingib-eraceae family is popularly known as white turmeric, zedoaria or gajutsu [8] This medicinal herb is largely found in East-Asian countries including Malaysia, Indo-nesia, China, India, Japan, Vietnam and Bangladesh [9] Traditionally, zedoaria is hugely consumed as a spice, a flavoring agent, a tonic, a treatment for menstrual dis-orders, vomiting, toothache and it is also made into per-fume [10, 11] A study done by Angel et al [12] reveals that zedoaria plants have a certain camphoraceous aroma and enormous functional active compounds such as essential oils, phenolics, and flavonoids which are strong

Srivastava et  al [13] reported that Curcuma zedoaria

Open Access

*Correspondence: ssalwaag@upm.edu.my; ssalwa.abdgani@gmail.com

1 Department of Agriculture Technology, Faculty of Agriculture, Universiti

Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia

Full list of author information is available at the end of the article

is closely related to Curcuma longa Therefore, the

cor-relative isolated active compounds found in zedoaria

such as curcumin, demethoxycurcumin and

bisdemeth-oxycurcumin could be effectively used as antioxidant

and anti-inflammatory, similar to Curcuma longa which

is popularly used as antioxidant, antiulcer,

anti-inflam-matory, etc Moreover, in vivo studies reported that the

rhizomes of the plant possess potent antioxidant activity

which exhibited higher radical scavenging activity [14]

The extraction of antioxidant compounds is a crucial

process to determine the quantity and type of

bioac-tive compounds, each with different therapeutic

prop-erties that will be extracted out According to Aybastier

et  al [15] many factors contribute to the efficiency of

extractions such as the type of solvent, the

concentra-tion of solvent, temperature, time, pH and solid–liquid

ratios Response surface methodology (RSM) is a

power-ful mathematical technique being widely used in many

industries for technological operations to optimize the

experimental conditions RSM is also useful to maximize

or minimize various independent variables as it evaluates

the effects of multiple factors and their respective

inter-actions on one or more response variables

simultane-ously Besides, RSM not only serves as a visual aid to have

a clearer picture about the effects of various factors on

extraction but also helps to locate the region where the

extraction is optimized

Therefore, the optimum extraction conditions (time, temperature and solvent ratio) to obtain the highest

amount of phenolic and flavonoid compounds from

Cur-cuma zedoaria leaves was identified using RSM

tech-nique Despite numerous studies on rhizomes of zedoary which investigated its antioxidant activity, the leaves

of the plant literally have not gained enough recogni-tion and study to the best of our knowledge In addirecogni-tion, Chanda and Nagani [16] reported that leaves, in general, are selected for the evaluation of total antioxidants activ-ity due to high content of bioactive compounds

Results and discussion Fitting the response surface models

A full factorial, central composite design (CCD) was used to identify the relationship between the response functions and process variables as well as to find out the conditions that optimized the extraction process The experimental design and corresponding three response variables are presented in Table 1 In the present study, according to the sequential model sum of squares, the highest order polynomials were utilized to select the models where the additional coefficients estimates were significant and the models are not aliased Hence, for all three independent variables and responses, a quadratic polynomial model was selected and fitted well as sug-gested by the software

Table 1 The experimental data obtained for the three responses based on the CCD matrix

Run no Type Temperature (X 1) Time (X 2) Solvent ratio (X 3) Phenolic

content mg/g GAE Flavonoid content mg QE/g extract

The final empirical regression model of their

relation-ship between responses and the three tested variables for

phenolic and flavonoid contents could be expressed by

the following quadratic polynomial equation [Eqs. (1–2)]:

(1)

Phenolic content = 122.36 + 5.74X1+ 2.03X2+ 4.10X3

− 4.11X1X2− 1.62X1X3− 2.77X2X3 + 1.34X2− 1.19X2− 3.55X2

(2)

Flavonoid content = 6.23 − 0.013X1− 0.016X2+ 0.091X3

− 0.08X1X2− 0.064X1X3+ 0.039X2X3

× 0.05X2+ 0.021X2− 0.070X2

where X 1 is the temperature, X 2 is the time and X 3 is the ethanol concentration ratio A negative sign in each equation represents an antagonistic effect of the variables and a positive sign represents a synergistic effect of the variables

The RSM model coefficients were validated by analysis

of variance (ANOVA) of the response variables for the quadratic polynomial model summarized in Table 2 The ANOVA results were calculated based on 95% confidence intervals and this analysis was crucial to determine the best fitted quadratic model for three independent

vari-ables A regression model was evaluated by using F

sta-tistics and lack of fit test Based on the results, it showed

Table 2 Analysis of variance (ANOVA) for the model

Sources Sum of squares Degree of freedom Mean squares F-value p-value

Phenolic content (mg/g GAE)

R 2 = 0.9993

Adj R 2 = 0.9987

CV% = 0.24

Flavonoid content (mg QE/g of extract)

R 2 = 0.9952

Adj R 2 = 0.9909

CV% = 0.17

that the model is highly significant when the computed

F-value is greater than the tabulated F-value and the

probability value is low (p  < 0.0001) indicating that the

individual terms in each response model were significant

on the interaction effect

The performance of the models was also checked by

calculating the determination coefficients R2, adjusted

R2, regression (p value), regression (F-value), lack of fit

(p-value), coefficient variation (C.V%) and probability

values related to the effect of the three independent

vari-ables Based on the result, the coefficient of

determina-tion R2 is defined as the ratio of the explained variation

to the total variation in total phenolic and total flavonoid

contents were R2 = 0.9993 and R2 = 0.9952 respectively

showing a good fit model The closer R2 value to unity,

the better and significant empirical model fits the actual

data Furthermore, the calculated adjusted R2 values for

studied responses variables were higher than 0.80, hence

there is a close agreement between the experimental

results and the theoretical values predicted by the

pro-posed models The coefficients of variations (C.V) for

total phenolic and flavonoid contents were 0.24 and 0.17

respectively, which indicates that a relatively lower value

of CV showed a better reliability of the response model It

was observed that the lack of fit gave no indication of

sig-nificance (p < 0.05) for all the models tested, thus proving

that the satisfactory fitness of the response surface model

was within the chosen range and significant (p < 0.05) to

the factors effect

Based on analysis of ANOVA, any terms from

quad-ratic polynomial coefficients model, large F-values and

a small p-values indicated a more significant effect on

the respective response variables The 3-D surface plots

of the fitted polynomial regression equations were

gen-erated by the software to better visualize the interaction

effect of independent variables on responses

Response surface analysis

Temperature, time and ethanol concentration are the main

factors that affect the extraction condition of the

maxi-mum total phenolics and flavonoids content This section

discusses how these conditions work on natural

antioxi-dants extraction Three-dimensional model graphs were

plotted as shown in the respective figures The response

surface plots of the model were done by varying two

vari-ables, within experimental range under investigation and

holding the other variables at its central level (0 levels)

Effects of process variables on the total phenolics content

(TP)

The amount of extracted phenolics content from

Cur-cuma zedoaria leaves ranged from 98 to 135 sample

extract, measured as gallic acid equivalent (GAE) The

value of mean recorded was 120.04  mg/g GAE of total leaves extracts The highest TP content was reported

at experimental run no 8 while the lowest TP content was observed at experimental run no 13 The ANOVA

showed the model F value of 1662.76 with probability

(p < 0.0001) which implies that the model is significant

and there is only 0.01% chances that this large F value

could occur due to noise Phenolic content was

signifi-cantly influenced at (p < 0.05) by all three linear (X 1 , X 2 ,

X 3 ), interaction parameters (X 1 X 2 , X 1 X 3 , X 2 X 3) and

quad-ratic parameters (X 1 2 , X 2 2 , X 3 2) (Table 2) The effect of their variables and their interaction on the responses can be seen in Fig. 1a–c

The surface plot in Fig. 1a demonstrates the function

of temperature (X 1 ) versus time (X 2) effect on total phe-nolic contents at fixed ethanol concentration (80:20) It was observed that increasing the extraction temperature

and time resulted in higher phenolic content in Curcuma

zedoaria leaves The maximum amount of phenolics can

be achieved at the highest temperature of 75–80  °C at the shortest extraction time of 80–100  min Neverthe-less, when the temperature was kept at the highest level

of 80 °C with longer extraction time at 120 min, they did not show any significant improvement in TP extraction

as the value continuously dropped This agreed with the working high temperature employed in this study which required short periods of time to avoid the degradation

of the phenolic compounds At short periods of time, the temperature enhanced the extraction process but for relatively long periods, the effect is inverted and the phenolic compounds are threatened by oxidation or deg-radation [17] Moreover, according to Vajić et al [18] pro-longed time of extraction enhances phenolic solubility due to Fick’s second law of diffusion which predicts that equilibrium of extraction will be achieved after a certain time These results are similar to a study reported by of Rajha et al [19] which showed the total phenolics from grape byproducts increased with the increment of tem-perature and reduction of time

Figure 1b depicts the effects of temperature (X 1)

ver-sus ethanol concentration (X 3) at constant extraction time 100  min The surface plot reveals that the maxi-mum phenolic content can be achieved at highest etha-nol concentrations (90:10) as compared with low ethaetha-nol concentrations (70:30) at fixed extraction temperature The higher phenolic content could be explained by the natural polarity of the solvents used [20] Ethanol and water were used in this study because they are safer to handle as compared to other organic solvents and more importantly, they are acceptable for human consump-tion Samuagam et al [21] stated that a suitable solvent ratio is able to improve the efficiency of extraction The surface plots also reveal that by increasing the extraction

Fig 1 Response surface plots for the effects of temperature, time and ethanol concentration on total phenolic contents of Curcuma zedoaria leaves

extracts a Temperature versus time b Ethanol concentration versus temperature c Time versus ethanol concentration

temperature to higher levels, the amounts of phenolic

gradually dropped and this might be explained by the

fact that the final equilibrium between the solvent

con-centrations in the plant matrix and the temperature will

be achieved after a certain concentration level [22] This

phenomenon is similar to a phenolic study from lettuce

by-products which can be explained by the use of higher

temperature and adequate solvent concentrations which

may cause softening of plant tissue, resulting in enhanced

diffusion rate and increase in the production of phenolic

compounds However, after a certain level, it will

subse-quently decline and remain constant as the extraction has

completed and they have achieved their equilibrium state

[23] Therefore, the maximum total phenolic content in

Curcuma zedoaria leaves can be obtained with optimum

ethanol concentration and an extraction temperature of

approximately 80–85 v/v% and 75–80 °C respectively

The response surface plot as a function of time (X 2)

versus ethanol concentration (X 3) at constant

tem-perature 70 °C is presented in Fig. 1c The surface plots

revealed that the higher TP contents can be obtained

when conducted at increasing ethanol concentration

at fixed extraction time Based on the result at constant

extraction time of 120  min, 90% of ethanol

concentra-tions yielded the most TP as compared with 70% ethanol

concentrations However, longer extraction time degrade

the phenolic activity in Curcuma zedoaria leaves

There-fore the optimum extraction of phenolic can be obtained

when conducted at a range of 80–90 v/v% and 100 min of

ethanol concentrations and extraction time respectively

Beyond this optimal, the TP content declined These

overall results of phenolic content indicate a similar trend

as observed in the phenolic content of tea (camellia

sin-ensis L.) fruit peel by Xu et al [24] where the TP contents

increased with increasing the independent variables

eth-anol concentration and processing time until a maximum

amount of phenolic was reached, thereafter, the amount

subsequently declined rapidly as reaction has completed

Effects of process variables on the total flavonoids content

(TF)

The mean experimental data showing the total

flavo-noid content from Curcuma zedoaria leaves at various

extraction conditions was 6.20 mg QE/g of extract in the

total range of 6.00–6.38 mg QE/g of extract The highest

content of TF was observed at experimental run no 17

meanwhile the lowest yield of TF was observed in

experi-mental run no 2 The ANOVA showed the model F value

of 229.66 with probability (p < 0.0001) which implies that

the model is significant and there is only 0.01% chances

that this large F value could occur due to noise

Flavo-noids content was significantly influenced at (p  <  0.05)

by all three linear (X 1 , X 2 , X 3), interaction parameters

(X 1 X 2 , X 1 X 3 , X 2 X 3 ) and quadratic parameters (X 1 2 , X 2 2 , X 3 2) (Table 2) The effect of their variables and their interac-tion on the responses can be seen in Fig. 2a–c

The 3D shows the response surface plot as a function of

temperature (X 1 ) versus time (X 2) at fixed extraction eth-anol concentration (80:20) as shown in Fig. 2a Response surface plot showed that extraction temperature exhib-ited a weaker effect whereas extraction time represented

a relatively significant effect on the flavonoids yield An increase in the yield of flavonoid could be significantly achieved with the increase of extraction time, at any level of extraction temperature Therefore, the opti-mum amount of flavonoid was achieved in this study at 65–70 °C and 90–100 min of extraction time However, the results of the present research for time and tempera-ture were different compared with other studies [4 19] This difference could be the due to differences in the type

of material, considering some plants may synthesize and accumulate the different amount of secondary metabo-lites (flavonoids) and also the optimization extractions range used in the study

The 3D surface plots in Fig. 2b shows the interaction

between extraction temperature (X 1) and ethanol

con-centration (X 3) at the fixed 100  min Statistical

analy-sis reveals that the most significant with p  <  0.0001 in

TF was ethanol concentration According to Bazykina

et al [25] flavonoids and their glycosides are thought to

be efficiently extracted from plant materials by ethanol solvent It was observed that the value of TF increased when ethanol concentration was increased from 70 to

90 v/v% at fixed 60  °C extraction temperature In con-trast, increasing the extraction temperature at highest ethanol concentrations resulted to decreased, TF values This phenomenon can be explained by the higher move-ment of the particles which causes plant tissue to rupture and hence allowing higher solubility of solvent until it starts to degrade to a lower value as it had achieved the stable state [26] The results obtained for flavonoids are

in agreement with the previous report from

Cryptotae-nia japonica hassk by Lu et al [27] where the flavonoid content increased when the temperature of extraction increased to below 70  °C and exhibited a decreasing trend above the optimum level of temperature Thus, as mentioned earlier the optimum extraction temperature for maximum flavonoid content was at 65–70  °C with 85–90 v/v% ethanol concentrations

Figure 2c illustrates the response surface plot between

the extraction time (X 2 ) and ethanol concentration (X 3)

at constant extraction temperature (70 °C) The response surface plots demonstrated that the value of TF obtained

in Curcuma zedoaria leaves mainly depended upon

ethanol concentrations An increase in ethanol concen-tration promoted the breakdown of the cell membrane

Fig 2 Response surface plots for the effects of temperature, time and ethanol concentration on total flavonoid content of Curcuma zedoaria leaves

extracts a Temperature versus time b Ethanol concentration versus temperature c Time versus ethanol concentration

that enhanced the permeability of the solvent into a solid

matrix In this study, highest flavonoids content can be

achieved when conducted at highest ethanol to water

ratio (90:10) as compared with (70:30) with increasing

extraction time A great increase in the yield also resulted

when extraction time was increased in the range of

80–120 min However, the time curve started to level off

at 100 min, which indicated that 100 min were required

to achieve maximum flavonoids activity

Optimization of extracting parameters and validation

of the model

In this study, the aim was to find the conditions which

gave the maximum yield of total phenolic and flavonoids

content The final result for the simultaneous

optimiza-tion using the desirability funcoptimiza-tion approach suggested

that the optimal ethanolic extraction conditions for

Cur-cuma zedoaria leaves extract were at 75 °C with 92 min

and 90:10 of ethanol concentration to achieve the best

combination for highest total phenolic and flavonoids

content These optimum extraction conditions were

eval-uated by considering the simultaneous response surface

and contour plot from the interaction between the

inde-pendent variables and responses of interest In order to

verify the optimum conditions, the Curcuma zedoaria

leaves were subjected using the optimal conditions above

and the results were statistically compared to the

pre-dicted values given by the design expert 7.0.0 software

of the response surface methodological (RSM) model

Based on the results, the predicted values of responses

were found to be quite comparable with experimental

values at 95% confidence level in Table 3

Conclusions

Response surface methodology (RSM) and a design

called central composite design (CCD) were successfully

developed to determine the optimum process parameters

and the second order polynomial models for

predict-ing responses were obtained The best combination of

extraction temperature, time and ethanol concentrations

were found to be 75  °C with 92  min and 90:10 ethanol

to water ratio which rendered a mean phenolic content

of 125.75 ± 0.17 mg/g GAE and 6.12 ± 0.23 mg/g QE of total flavonoid content from experimental run and thus indicated good antioxidant activities from the leaves of

Curcuma zedoaria.

Materials and methods Raw materials

Curcuma zedoaria leaves were collected from a local

farmer in Kedah, Malaysia The chemicals, sodium car-bonate, aluminium chloride, ethanol was purchased from

J Kollin Chemicals, Germany Folin-Ciocalteu’s phenol reagent, gallic acid and quercetin were purchased from Sigma-Aldrich (St Louis, MO, USA) All other chemical reagents used in this study were of analytical grade class

Plant extraction

The air-dried leaves of Curcuma zedoaria plant were

cut into pieces and ground into powder form using a mechanical blender About 0.5 g of powdered leaves were exactly weighed into a 150 mL round bottomed flask and mixed with ethanol The extraction process was per-formed using a reflux systems equipped with a tempera-ture controller and digital timer The extract was then filtered through normal filtration using Whatman filter paper and vacuum-dried in a rotary evaporator, at 40 °C until the excess solvent was completely removed

Experimental design

The optimization of the extraction conditions from

the Curcuma zedoaria leaves was established by using

response surface methodology (RSM) This power-ful mathematical and statistical technique is usepower-ful for modeling and analysis of problems in which a response

is influenced by several independent variables and the objective is to find the relationship between the factor and the response to optimize the conditions A design expert software Version 7.0.0, (Stat ease Inc., Minne-apolis, USA) was used in this study The experimen-tal plan was carried out based on three factor/five level design referred to as rotatable central composite design (CCD) The selection of CCD as the experimental design

is because it is more precise for estimating factor effects [28] Hence, the interaction effect between factors can be evaluated and optimized in the full factor space

The design consisted of twenty experimental runs, including six replicates at the center points The center points were utilized to define the experimental error and the reproducibility of the data The independent variables

in this study were extraction temperature (X 1: 60–80 °C),

time (X 2 : 80–120  min) and ethanol concentrations (X 3: 70–90% v/v ethanol/water) The five levels of values for the independent variables were explicit of their coded and uncoded forms in Table 4 The value of independent

Table 3 Comparison between  the predicted and 

experi-mental values for  antioxidants from  extracts of  Curcuma

zedoaria leaves

Condition Response values

Phenolic content mg/g GAE Flavonoid content mg/g QE

Experimental 125.75 ± 0.17 6.12 ± 0.23

variables was expressed in their coded values as −1, 0,

+1 interval shows the low, center, and high level of each

variable, respectively The multiple regression analysis

was performed on the data of response variables such as

total phenols and flavonoid content obtained as affected

by the extraction conditions and was fitted into a

polyno-mial regression equation as shown in the following

equa-tions (Eq. 3);

where Y represents the response variables to be modeled;

β o is a constant, βi,βii and βij are the linear, quadratic and

cross-product coefficients, respectively X i and X j are the

levels of the independent variables k is the number of

variables and e is the random error of the model.

Determination of total phenolic content

The total phenolic compounds in Curcuma zedoaria

leaves was developed using the method of Singleton and

Rossi [29] with minor modifications For each sample,

100 μL (1 mg/mL) of the sample extract was mixed with

50 μL Folin-Ciocalteu’s reagent (2 N) previously diluted

with 7.9  mL distilled water After 4  min, 1.5  mL of 7.5

w/v% sodium carbonate solution was added to the

mix-ture and incubated in the dark room at room

tempera-ture for 2  h The absorbance values of the sample were

measured at 765 nm using a UV–VIS microplate reader

Standard of gallic acid with different concentrations

(25–1000  μg/L) was prepared in this study to generate

a standard calibration curve The samples were

calcu-lated based on the standard calibration curve and were

expressed as mg gallic acid equivalent (mg/g GAE)

Determination total flavonoids content

The content of flavonoid in the studied leaves extract

was determined using spectrophotometric method [30]

From each sample, 100 μL (1 mg/mL) were mixed with

2% AlCl3 and incubated for 15 min at room temperature

(3)

Y = βo+

k

i=1

βiXi+

k

i=1

βiiXi2+

k

i=1

k

ji

βijXiXj+e

The absorbance was measured at λ  =  406  nm The same procedure was repeated for the standard solution

of quercetin at different concentrations (25–250  μg/ mL) and the calibration line was obtained Based on the measured absorbance, the concentration of flavo-noids was calculate (mg/mL) on the calibration line and the content of flavonoids in extracts was expressed in terms of quercetin equivalent, QE (mg of quercetin/g of extract)

Statistical analysis and optimization

Best fitted model of response can be achieved by high-lighting these statistical parameters including the

adjusted multiple correlation coefficients (adjusted R 2),

multiple correlation coefficients (R2), coefficient

varia-tion (C.V%), lack of fit, regression F-value and regres-sion p-value by using analysis of variance (ANOVA) This

statistical approach was used to summarize the results obtained under all experimental conditions with a con-fidence interval of 95% set to test the significant effect

of the factors and their interaction The optimal extrac-tion condiextrac-tions were selected based on the condiextrac-tion

of achieving the highest total phenolics and flavonoids

content in Curcuma zedoaria leaves by using the

desir-ability function approach in design expert software The fitted polynomial equation was expressed in the form of three-dimensional surface plots in order to illustrate the relationship between responses and the experimental variables used

Verification of models

The optimal conditions for the extraction of the total

phenolic and flavonoid content from Curcuma zedoaria

leaves, in terms of extraction temperature, time and ethanol concentrations, were determined by com-paring the actual experimental values with predicted value from the final response regression equations Besides, a few random extraction conditions were pre-pared in order to validate the models This action is of utmost importance to confirm the adequacy of the final reduced models

Table 4 Independent test variables and their coded and uncoded value used for CCD matrix

Variables Units Coded & uncoded level of variables

Solvent ratio, X 3

Authors’ contributions

NFA participated in the design of the study and performed the statistical

analysis SSAG and NFMM participated in the sequence alignment and drafted

the manuscript All authors read and approved the final manuscript.

Author details

1 Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra

Malaysia (UPM), 43400 Serdang, Selangor, Malaysia 2 Halal Products Research

Institute, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia

3 Institute for Mathematical Research (INSPEM), Universiti Putra Malaysia

(UPM), 43400 Serdang, Selangor, Malaysia

Acknowledgements

The authors gratefully acknowledge the financial support from a Graduate

Research Fellowship (GRF) under UPM for the scholarship.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in

pub-lished maps and institutional affiliations.

Received: 24 August 2016 Accepted: 21 September 2017

References

1 Ghasemzadeh A, Jaafar HZE, Rahmat A (2010) Antioxidant activities, total

phenolics and flavonoids content in two varieties of malaysia young

ginger (Zingiber officinale Roscoe) Molecules 15:4324–4333

2 Karabegović IT, Stojičević SS, Veličković DT, Todorović ZB, Nikolić NČ, Lazić

ML (2014) The effect of different extraction techniques on the

composi-tion and antioxidant activity of cherry laurel (Prunus laurocerasus) leaf and

fruit extracts Ind Crops Prod 54:142–148

3 Heydari Majd M, Rajaei A, Salar Bashi D, Mortazavi SA, Bolourian S (2014)

Optimization of ultrasonic-assisted extraction of phenolic compounds

from bovine pennyroyal (Phlomidoschema parviflorum) leaves using

response surface methodology Ind Crops Prod 57:195–202

4 Sheng ZL, Wan PF, Dong CL, Li YH (2013) Optimization of total flavonoids

content extracted from Flos Populi using response surface methodology

Ind Crops Prod 43:778–786

5 Liu Y, Wei S, Liao M (2013) Optimization of ultrasonic extraction of

phe-nolic compounds from Euryale ferox seed shells using response surface

methodology Ind Crops Prod 49:837–843

6 Huo L, Lu R, Li P, Liao Y, Chen R (2011) Antioxidant activity, total phenolic

and total flavonoid of extracts from the stems of Jasminum nervosum

Lour Grasas Aceites 2:149–154

7 Kalita P, Barman TK, Pal TK, Kalita R (2013) Estimation of total flavonoids

content (TFC) and antioxidant activities of methanolic whole plant

extract of biophytum sensitivum linn J drug deliv Ther 4:33–37

8 Lobo R, Prabhu KS, Shirwaikar A, Shirwaikar A (2009) Curcuma zedoaria

Rosc (white turmeric): a review of its chemical, pharmacological and

ethnomedicinal properties J Pharm Pharmacol 61:13–21

9 Tholkappiyavathi K, Selvan KM, Neyanila SK, Yoganandam GP, Gopal V

(2013) A concise review on Curcuma Zedoaria Int J Phyther 2:1–4

10 Srivastiva NR, Lal BR, Kumar SV, Dhirendra K (2013) Physicochemical

and phytochemical investigation of three different species of Curcuma

rhizome Int Res J Pharm 4:163–166

11 Anisuzzaman M, Sharmin SA, Mondal SC, Sultana R, Khalekuzzaman M

(2008) In vitro microrhizome induction in Curcuma zedoaria (christm.)

roscoe—a conversation prioritized medicinal plant J Biol Sci 8:1216–1220

12 Angel GR, Vimala B, Nambisan B (2012) Phenolic content and antioxidant

activity in five underutilized starchy Curcuma species Int J Pharmacogn

Phytochem Res 4:69–73

13 Srivastava S, Mehrotra S, Rawat A (2011) Pharmacognostic evaluation of

the rhizomes of Curcuma zedoaria Rosc Pharmacogn J 3:18–24

14 Himaja M, Ranjitha A, Ramana MV, Anand M, Asif K (2010) Phytochemical

screening and antioxidant activity of rhizome part of Curcuma Zedoaria

Int J Res Ayurveda Pharm 1:414–417

15 Aybastier O, Isik E, Sahin S, Demir C (2013) Optimization of ultrasonic-assisted extraction of antioxidant compounds from blackberry leaves using response surface methodology Ind Crop Prods 44:558–565

16 Chanda SV, Nagani KV (2010) Antioxidant capacity of Manilkara zapota L

leaves extracts evaluated by four in vitro methods Nat Sci 8:260–266

17 Yilmaz Y, Toledo RT (2006) Oxygen radical absorbance capacities of grape/wine industry byproducts and effect of solvent type on extraction

of grape seed polyphenols J Food Compos Anal 19:41–48

18 Vaji ć UJ, Grujić-Milanović J, Živković J, Šavikin K, Gođevac D, Miloradović

Z, Bugarski B, Mihailović-Stanojević N (2015) Optimization of extraction

of stinging nettle leaf phenolic compounds using response surface methodology Ind Crops Prod 74:912–917

19 Rajha HN, Darra NE, Hobaika Z, Boussetta N, Vorobiev E, Maraoun RG, Louka N (2014) Extraction of total phenolic compound, flavonoids, antho-cyanins, and tannis from grape byproducts by response surface method-ology Influence of solid-liquid ratio, particle size, time, temperature and solvent mixtures on the optimization process Food Nutr Sci 5:397–409

20 Tan MC, Tan CP, Ho CW (2013) Effects of extraction solvent system, time

and temperature on total phenolic content of henna (Lawsonia inermis)

stems Int Food Res J 20:3117–3123

21 Samuagam L, Sia CM, Akowuah GA, Okechukwu PN, Yim HS (2013) The effect of extraction conditions on total phenolic content and free radi-cal scavenging capacity of selected tropiradi-cal fruits’ peel Heal Environ J 4:80–102

22 Sai-Ut S, Benjakul S, Kraithong S, Rawdkuen S (2015) Optimization of anti-oxidants and tyrosinase inhibitory activity in mango peels using response surface methodology LWT Food Sci Technol 64:742–749

23 Gomes T, Delgado T, Ferreira A, Pereira JA, Baptista P, Casal S, Ramalhosa

E (2013) Application of response surface methodology for obtaining

lettuce (Lactuca sativa L.) by-products extracts with high antioxidative

properties Ind Crops Prod 44:622–629

24 Xu P, Bao J, Gao J, Zhou T, Wang Y (2012) Optimization of extraction of

phenolic antioxidants from tea (camellia sinensis L.) fruit peel biomass

using response surface methodology BioResources 2:2431–2443

25 Bazykina NI, Nikolaevskii AN, Filippenko TA, Kolerva VG (2002) Optimiza-tion of condiOptimiza-tions for the extracOptimiza-tion of natural antioxidants from raw plant materials Pharm Chem J 36:46–49

26 Yoswathana N (2013) Optimization of ScCO2 extraction of rambutan seed oil using response surface methodology Int J Chem Eng Appl 4:187–190

27 Lu J, Zhou C, Rong O, Xu Y (2013) Optimization of microwave-assisted

extraction of flavonoids from Cryptotaenia japonica hassk using response

surface methodology Adv J food Sci Technol 5:310–317

28 Mohd Taib SH, Abd Gani SS, Ab Rahman MZ, Basri M, Ismail A, Shamsudin

R (2015) Formulation and process optimizations of nano-cosmeceuticals containing purified swiftlet nest RSC Adv 5:42322–42328

29 Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phos-phomolybdic-phosphotungstic acid reagent Am J Enol Vitic 16:144–153

30 Stankovic M (2011) Total phenolic content, flavonoid concentration and

antioxidant activity of Marrubium peregrinum L extracts Kragujevac J Sci

33:63–72